CN105624128B

CN105624128B - Immobilized monoamine oxidase and application thereof in synthesis of chiral azabicyclo compound

Info

Publication number: CN105624128B
Application number: CN201410697504.3A
Authority: CN
Inventors: 罗煜; 丁时澄; 瞿旭东; 钱龙
Original assignee: Abiochem Biotechnology Co Ltd
Current assignee: Yikelai Biotechnology Group Co ltd
Priority date: 2014-11-26
Filing date: 2014-11-26
Publication date: 2020-03-17
Anticipated expiration: 2034-11-26
Also published as: CN105624128A

Abstract

The invention provides an immobilized monoamine oxidase with firm combination and less enzyme activity loss, which is used for synthesizing (1S,2S,5R) -aza-dicyclic compounds through enzyme catalysis. Compared with other methods, the immobilized enzyme prepared by the invention has the advantages of firm binding, less enzyme activity loss, simple separation, more times of repeated utilization, low cost of the oxidation-addition process, mild reaction conditions, environmental friendliness, simple and convenient operation and easy industrial amplification, thereby having good industrial application prospect.

Description

Immobilized monoamine oxidase and application thereof in synthesis of chiral azabicyclo compound

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to immobilized monoamine oxidase, a preparation method of the immobilized monoamine oxidase, and application of the immobilized monoamine oxidase as a catalyst in asymmetric synthesis of a chiral azabicyclo compound.

Background

The enzyme is a biological macromolecular substance with a catalytic function produced by organisms, and can catalyze various organic chemical reactions under the mild conditions of normal temperature and pressure as a biocatalyst. The enzyme catalysis has high efficiency which is 10 times higher than that of the common catalyst⁷～10¹³Doubling; the enzyme catalysis has high specificity, and can reduce or avoid side reaction to obtain a product with high purity; the enzyme catalysis reaction also has high stereoselectivity and regioselectivity, and does not need protection and deprotection. These advantages of enzymes have greatly facilitated research into the use of enzymes and enzyme technology. However, since enzymes are mostly proteins, their high-order structures are very sensitive to the environment, and various factors such as physical factors (temperature, pressure, electromagnetic field, etc.), chemical factors (oxidation, reduction, organic solvent, metal ions, ionic strength, pH, etc.), and biological factors (enzyme modification and enzyme degradation, etc.) may lose biological activity. Even under the optimum conditions, the enzyme is gradually inactivated, and the reaction time is prolongedThe speed will gradually decrease; in addition, the enzyme cannot be recovered after the reaction and can only be produced by a batch process, which is costly for the modern biocatalysis industry.

In order to solve the above problems, an immobilized enzyme has been designed, which is bound in a special medium so that it is separated from the reaction system but can still exchange molecules with the substrate. The immobilized enzyme has the same catalytic property as common solid chemical catalyst, and has the advantages of recovery, repeated use and the like, and the production process can realize continuous and automatic production.

The immobilization method of the immobilized enzyme comprises an adsorption method, an embedding method, a crosslinking method and a covalent binding method. The adsorption method can be divided into a physical adsorption method and an ion adsorption method, and has the advantages of mild conditions, less enzyme activity loss, easy regeneration, loose fixation and easy desorption. The embedding method mainly adopts calcium alginate, carrageenan, polyacrylamide, nylon membrane or nitrocellulose microcapsule for embedding, and the method is generally mild, has little loss of enzyme activity, but has large mass transfer resistance and is not beneficial to the catalysis of substrates with larger molecules or low solubility. Glutaraldehyde crosslinking is used most in the crosslinking method, so that the operation is simple and convenient generally, but the reaction is violent, and the enzyme activity loss is large; and the mechanical property is poor, the granularity is fine, and the separation is difficult. The covalent bonding method is commonly used for diazotization reaction of arylamine carriers, cyanogen bromide-imine carbonate reaction of hydroxyl carriers, carbodiimine reaction of carboxyl carriers, disulfide bond exchange reaction of sulfhydryl carriers and the like, and is the most widely researched and applied method at present due to the characteristics of firm bonding, good stability and the like. However, the method has harsh conditions, violent reaction and large enzyme activity loss (generally, the residual enzyme activity is about 30 percent).

Boceprevir and telaprevir are Hepatitis C Virus (HCV) protease inhibitors having the following chemical structures:

phase iii SPRINT-2 studies have shown that 24 weeks of standard treatment with boceprevir can increase the SVR rate in patients initially treated for HCV genotype 1 compared to standard treatment. The antiviral efficacy of the boceprevir standard treatment for 24 weeks is similar to that of the boceprevir standard treatment for 44 weeks. Fragment A is one of key intermediates for synthesizing the boceprevir, and the structure of fragment A is as follows:

the following synthetic routes are currently predominant for fragment a:

route 1: WO2007075790 discloses the utilization of 6, 6-dimethyl-3-oxabicyclo [3.1.0] hexane-2, 4-dione to lactamize, reduce carbonyl, reduce and add cyano, hydrolyze, split, etc. to obtain the salt of fragment a, the specific reaction route is as follows:

the cyano addition in the route utilizes silver nitrate, potassium cyanide and hydrochloric acid, so that the cost is high, great difficulty is brought to post-treatment and three-waste treatment, and the method is not beneficial to industrial production.

Route 2: the literature (Journal of Medicinal Chemistry,2006, Vol.49, No.20, 6074-:

because the reduction reaction in the route adopts lithium aluminum hydride and palladium carbon, the reaction conditions are harsh, the post-treatment is more complicated, and the method is not beneficial to industrial production.

Route 3: WO2004113295 discloses a hydrochloride of fragment A obtained by using 6, 6-dimethyl-3-oxabicyclo [3.1.0] hexane-2, 4-diketone as a raw material and performing alcoholysis, amidation, amide group and ester group reduction, oxidation of alcohol into aldehyde after amino protection, cyclization, cyanation, hydrolysis and deprotection, wherein the specific reaction route is as follows:

although the route has the advantages of cheap and easily-obtained starting materials, the route needs to adopt a secondary reduction reaction when preparing (1R,3S) -3-aminomethyl-2, 2-dimethyl cyclopropane methanol, namely, ester groups are reduced by utilizing lithium borohydride or sodium borohydride selected from alane and in the presence of trimethylsilyl chloride, and amide groups are reduced by utilizing lithium aluminum hydride or sodium triacetoxyborohydride, and the reduction reaction conditions are severe, the temperature requirement is harsh, the reaction time is long, the post-treatment is complicated, and the safety is low, so that the industrial application of the route is influenced.

Route 4: the literature (j.am. chem. soc.2012,134,6467-6472) discloses a process for the preparation of (1S,2S,5R) -6, 6-dimethyl-3-aza-bicyclo [3.1.0] hexane-2-carbonitrile using the elimination-addition reaction of monoamine oxidase from Aspergillus niger (Aspergillus niger), as follows:

the method uses monoamine oxidase to oxidize prochiral amine into chiral imine in NaHSO₃In the presence of a catalyst to form an addition product; the latter is reacted with NaCN to give a chiral imine addition product (as above), which is methanolyzed to give amino acid methyl ester hydrochloride. The method stereoselectively constructs 3 chiral centers, is simpler and more convenient than a chemical method, is easy to operate and is environment-friendly; however, the imine product is volatile and has a low flash point, and additional NaHSO is required₃The steps are relatively complicated.

The fragment B is one of key intermediates for synthesizing telaprevir, and the structure of the fragment B is as follows:

the current synthetic routes for fragment B are mainly as follows:

route 1: EP0600741 discloses the following route:

because the target compound synthesized by the route exists in the oily mixture, a pure product can be obtained only by silica gel column chromatography separation, the post-treatment is complicated, and the large-scale production cannot be realized.

Route 2: WO0218369 discloses the following route:

because the DIBAL-H reduction is adopted in the route, the reagent cost is higher, and the preparation condition is harsh (the reaction is required to be carried out at minus 78 ℃), the route is not suitable for the industrial production requirement.

Route 3: the following route is disclosed in CN 101463001:

the route uses the virulent cobalt carbonyl, so that the cost is too high, and the environment-friendly requirement cannot be met, so that the route cannot meet the industrial requirement.

Route 4: WO2008090819 discloses the following route:

the starting materials of the route are difficult to synthesize and cannot be purchased in batches, so the route is not suitable for the requirement of industrial production.

Disclosure of Invention

Aiming at the problems that monoamine oxidase is difficult to separate in the enzyme catalysis process, and the enzyme activity loss is large or the mass transfer is difficult in the enzyme immobilization process, the invention derivatizes commercial epoxy resin with iminodiacetic acid (IDA) and nickel sulfate in sequence to obtain an enzyme immobilization carrier, and then uses monoamine oxidase to perform immobilization. The immobilization method has mild reaction and basically no loss of enzyme activity. The immobilized enzyme and an oxidant are used for carrying out oxidation reaction on a substrate prochiral compound azabicyclic compound, and then the (1S,2S,5R) -azabicyclic compound is prepared by addition reaction with MR, can be repeatedly used for 5 batches, and is simple to separate.

One aspect of the present invention provides a recombinant monoamine oxidase encoded by the nucleotide sequence set forth in SEQ ID No. 2.

A method of making the recombinant monoamine oxidase described above, comprising: carrying out double digestion on a plasmid containing the monoamine oxidase gene shown in SEQ No.2 and a pET28a vector by using the same restriction enzymes Nde I and Xho I, recovering double digestion fragments, and connecting the fragments by using T4DNA ligase to form a recombinant expression plasmid pET28 a-BYK-MAON; the recombinant expression plasmid pET28a-BYK-MAON is transformed into Escherichia coli (E.coli) BL21(DE3), and the genetic engineering strain of the invention, namely E.coli BL21(DE3)/pET28a-BYK-MAON, can be obtained; inoculating the genetically engineered strain E.coliBL21(DE3)/pET28a-BYK-MAON into LB culture medium containing kanamycin, and culturing when the optical density OD of the culture solution₆₀₀When the concentration reaches 0.7-0.9, adding isopropyl- β -D-thiogalactopyranoside (IPTG) with the final concentration of 0.05-1.0 mmol/L for induction, and expressing the recombinant monoamine oxidase at the induction temperature of 10-40 ℃.

The second aspect of the invention provides an enzyme immobilization carrier.

Adding amino resin into ethylene glycol diglycidyl ether to form epoxy resin, placing into 20mM sodium phosphate buffer solution of 100mM iminodiacetic acid (IDA) and pH 7.0-10.0 (preferably pH7.5), reacting for 0.5-2 hr at 20-80 deg.C (preferably 60 deg.C), washing with water, and adding 40mM NiSO₄And (3) resuspending and filtering the solution, and washing the solution by using water to obtain the enzyme immobilized carrier.

In a particular embodiment, the amino resin according to the invention is an amino resin from Shanghai Huazheng, in particular an amino resin of type 355.

The invention also provides an immobilized monoamine oxidase.

The immobilized monoamine oxidase is obtained by immobilizing the recombinant monoamine oxidase of the present invention on the enzyme-immobilized carrier of the present invention.

Adding 1-10 times (preferably 5 times) of the immobilized carrier into 50mM sodium phosphate buffer solution with the pH value of 7.5 of the recombinant monoamine oxidase, stirring at room temperature for 2-6 hours, filtering and washing to obtain the immobilized enzyme. The enzyme activity of the obtained immobilized enzyme reaches 13U/g, and the recovery rate is 68.4%.

The invention also provides the use of a monoamine oxidase or immobilized monoamine oxidase of the invention for the catalysis of the oxidation-addition reaction of prochiral compounds to form chiral addition products.

In the above applications, the conditions of the oxidation-addition reaction can be selected according to the conditions conventional in the art for such reactions, and are preferably as follows:

the prochiral compound is an azabicyclo compound, namely a compound shown as a formula I:

wherein R1 and R2 are independently selected from H, C1-C5 alkyl; a is methylene; n is an integer of 0 to 5.

The prochiral azabicyclic compound is oxidized with an oxidant under the action of immobilized monoamine oxidase, and then is subjected to addition reaction with MR to generate a (1S,2S,5R) -aza-bicyclo compound, namely the compound shown in a formula II:

wherein the content of the first and second substances,

r1 and R2 are independently selected from H, C1 to C5 alkyl; a is methylene; n is an integer of 0 to 5;

r is selected from-CN, -SCN, -SO₃Na、-SO₃H；

M is selected from alkali metal, alkaline earth metal and H.

The conditions of the oxidation-addition reaction of the present invention may be selected according to the conditions conventional in such reactions in the art, and preferably, the application comprises the steps of: under the catalysis of the immobilized monoamine oxidase and catalase, the prochiral azabicyclic compound and MR are subjected to asymmetric oxidation-addition reaction with an oxidant in an aqueous solution with the pH of 6.0-8.0 to form an optical chirality pure substitution product.

Wherein the preferable concentration of the prochiral azabicyclic compound in the reaction liquid is 100-1000 mmol/L. The dosage of the immobilized monoamine oxidase of the invention is preferably 25-250 g/L. The amount of MR used is preferably 100 to 1200 mmol/L. The amount of catalase is a catalytically effective amount, preferably 0.01-0.1 g/L. The aqueous solution can be a buffer solution which is conventional in the field as long as the pH range is 6.0-8.0, and a phosphate buffer solution, such as a disodium hydrogen phosphate buffer solution, is preferred; the concentration of the phosphate buffer solution is preferably 0.05-0.1 mol/L, wherein the concentration refers to the total concentration of the conjugate acid and the conjugate base in the buffer solution. The oxidation-addition reaction is performed at room temperature, preferably 20 to 50 ℃, more preferably 20 to 35 ℃. The time of the oxidation-addition reaction is preferably based on a residual concentration of the substrate of less than 5%. After the oxidation-addition reaction is completed, (1S,2S,5R) -aza-bicyclo compound can be extracted from the reaction solution according to a method conventional in the art.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: aiming at the problems that monoamine oxidase is difficult to separate in the enzyme catalysis process and the reported problems of large enzyme activity loss or difficult mass transfer and the like in the enzyme immobilization process, the immobilized enzyme with firm combination and less enzyme activity loss is provided and is used for the enzymatic synthesis of the (1S,2S,5R) -aza-bicyclo compound. Compared with other methods, the immobilized enzyme prepared by the invention has the advantages of firm binding, less enzyme activity loss, simple separation, more times of repeated utilization, low cost of the oxidation-addition process, mild reaction conditions, environmental friendliness, simple and convenient operation and easy industrial amplification, thereby having good industrial application prospect.

Drawings

FIG. 1 is an agarose gel electrophoresis of the monoamine oxidase gene PCR product. M is DNA molecular weight standard, lane 1 is the monoamine oxidase gene amplified by PCR.

FIG. 2 is a polyacrylamide gel electrophoresis diagram of a crude enzyme solution of monoamine oxidase. M is a molecular weight standard, lane 1 is whole mycoprotein lysate.

Detailed Description

The invention is further illustrated by the following examples, but is not limited thereto. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

The material sources in the following examples are:

pET28a-BYK-MAON was constructed by this company.

Amino resin 335 was purchased from Shanghai Huazhen technology, Inc.

The pET28a expression plasmid, E.coli DH5 α and E.coli BL21(DE3) competent cells, 2 XTaq PCRMasterMix, agarose gel DNA recovery kit were purchased from Changsheng biotechnology, Limited liability company, Beijing ancient China.

EXAMPLE 1 preparation of immobilization Carrier

100g of amino resin with the model number of 335 from Shanghai Huazhen company is slowly added into 200mL of 5% (v/v) ethylene glycol diglycidyl ether toluene solution under stirring, the mixture is stirred and reacted for 1 hour at 60 ℃, then the mixture is washed by toluene and water, then the mixture is put into 350mL of 20mM sodium phosphate buffer solution (pH 7.5) containing 100mM iminodiacetic acid (IDA) and reacted for 2 hours at 60 ℃, the mixture is washed by water after being filtered, then the mixture is resuspended by 200mL of 40mM nickel sulfate solution, the mixture is stirred for 30 minutes at room temperature, and the mixture is filtered and washed by water to obtain the immobilized carrier.

Example 2 preparation of recombinant monoamine oxidase-expressing transformants

Constructing a recombinant expression vector pET28a-BYK-MAON containing monoamine oxidase BYK-MAON (the nucleotide sequence of ORF and the amino acid sequence coded by the nucleotide sequence are respectively shown as SEQ ID No.1 and SEQ ID No. 2) according to Chinese patent application No. 201410441595.4, transforming the recombinant expression plasmid into a competent cell of Escherichia coli (E.coli) DH5 α, carrying out heat shock for 45 seconds under the transformation condition, screening positive recombinants on a resistant plate containing kanamycin, picking up a monoclonal, carrying out colony PCR (polymerase chain reaction) verification on the positive clones, culturing the recombinant bacteria, extracting the plasmid after the plasmid is amplified, re-transforming the plasmid into a competent cell of E.coli BL21(DE3), coating a transformation solution on a LB plate containing kanamycin, carrying out inversion culture at 37 ℃ overnight, obtaining a positive recombinant transformant E.coli BL21(DE3)/pET28a-BYK-MAON, and carrying out PCR verification on the colony of the positive clones.

EXAMPLE 3 expression of recombinant monoamine oxidase

The recombinant E.coli BL21(DE3)/pET28a-BYK-MAON obtained in example 3 was inoculated into a kanamycin-containing LB medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.0), cultured overnight with shaking at 37 ℃ and 1% (v/v) of the inoculum size in a 500mL Erlenmeyer flask containing 200mL of LB medium, and cultured with shaking at 37 ℃ and 200rpm, when the OD of the culture solution is determined₆₀₀When the concentration reaches 0.8, IPTG with the final concentration of 0.1mmol/L is added as an inducer, after induction is carried out for 12h at 26 ℃, the culture solution is centrifuged, cells are collected and washed twice by normal saline, and the resting cells are obtained. Suspending the obtained resting cells in a buffer solution with the pH value of 7.5, carrying out ultrasonic disruption in an ice bath, centrifuging and collecting supernatant fluid, namely the crude enzyme solution of the recombinant monoamine oxidase. Protein concentration was determined by the Bradford method. The crude enzyme solution and the precipitate were analyzed by polyacrylamide gel electrophoresis, and the recombinant protein was present in a soluble form. And (3) freeze-drying the crude enzyme liquid by using a freeze dryer to obtain freeze-dried crude enzyme powder.

Example 4 preparation of immobilized enzyme

20g of the lyophilized crude enzyme powder of recombinant monoamine oxidase obtained in example 4 was resuspended in 2L of 50mM sodium phosphate buffer solution pH7.5, 100g of the immobilized carrier obtained in example 1 was added, stirred at room temperature for 4 hours, filtered, and washed with water to obtain an immobilized enzyme.

Example 5 measurement of Activity of free monoamine oxidase and immobilized enzyme

Benzaldehyde was used as a standard curve in the range of 0.1mM to 0.5 mM. The enzyme activity was defined as the amount of 1U of enzyme required for 1. mu. mol of benzaldehyde to be produced in 1 minute.

1mL of 5mM benzylamine solution (50mM sodium phosphate buffer, pH7.5) was added with 0.02g of immobilized enzyme or 100. mu.L of free enzyme, reacted at 30 ℃ for 10min, then added with 400. mu.L of DNPH, mixed well, added with 2400. mu.L of NaOH solution, reacted for 5min, and the absorbance was measured at 450 nm.

TABLE 1 enzyme Activity of free and immobilized enzymes

Numbering	Enzyme	Enzyme activity yield (%)	Specific activity (U/g)
				1	Free enzyme	100	19.6
2	Immobilized enzyme prepared from immobilized carrier	68.4％	13

Example 6 biocatalysis of immobilized monoamine oxidase

10g of immobilized monoamine oxidase was added to 20mL of phosphate buffer (50mmol/L, pH7.5), followed by slow dropwise addition of 16mL of 6, 6-bis-chiral compound containing 1.57mol/L of prochiral compoundMethyl-3-aza-bicyclo [3.1.0]Hexane and 2.25mol/LNaHSO₃To the phosphate buffer (50mmol/L, pH7.5), catalase was added to a final concentration of 0.1 g/L. Pure oxygen is introduced, the temperature is kept at 25 ℃, and the reaction is carried out for 24 hours. At the end of the reaction, the conversion and residual enzyme activity of the reaction were measured according to the method of example 5.

TABLE 2 conversion rate and residual enzyme activity of different batches of immobilized enzyme catalytic oxidation-addition reaction

Batches of	Reaction time (h)	Conversion rate	Residual enzyme activity
				1	24	>95％	98％
2	24	>95％	97％
				3	24	>95％	95％
4	24	93％	91％
				5	24	90％	89％

Examples 7-11 biocatalysis of immobilized enzymes

The conversion and residual enzyme activity of different batches of immobilized enzyme to catalyze the oxidation-addition reaction of various substrates was tested essentially as described in example 6 (see table 3).

TABLE 3 different batches of immobilized enzymes catalyze the conversion rate and residual enzyme activity of various substrate oxidation-addition reactions

EXAMPLE 12 esterification reaction

1.36g of (1S,3aR,6aS) -octahydrocyclopenta [ c ] pyrrole-1-carbonitrile is dissolved in 20mL of methanol, 15mL of a hydrogen chloride-methanol solution is dropwise added at 0-10 ℃, the mixture is stirred at room temperature for reaction for 2 hours, and after the reaction is finished, the solvent is evaporated under reduced pressure to obtain 1.91g of (1S,3aR,6aS) -octahydrocyclopenta [ c ] pyrrole-1-carboxylic acid methyl ester hydrochloride, wherein the yield is 92.3%.

Claims

1. An immobilized monoamine oxidase which is a monoamine oxidase which will be encoded by SEQ ID No: 2, and is characterized in that the enzyme immobilization carrier is obtained by derivatizing epoxy resin sequentially by iminodiacetic acid and nickel sulfate, wherein the epoxy resin is prepared by adding amino resin into ethylene glycol diglycidyl ether, and the amino resin is 335 amino resin of Shanghai Huasha science and technology Limited.

2. A process for preparing an immobilized monoamine oxidase of claim 1 comprising the steps of:

(1) will be represented by SEQ ID No: 2, resuspending the monoamine oxidase encoded by the amino acid sequence shown in fig. 2 in a 50mM sodium phosphate buffer solution at pH7.5, adding 5 times the weight of the enzyme-immobilized carrier defined in claim 1, and stirring at room temperature for 2 to 6 hours;

(2) and (3) filtering and washing the immobilized carrier reacted in the step (1), namely the immobilized monoamine oxidase.

3. Use of an immobilized monoamine oxidase according to claim 1 or prepared according to the process of claim 2 for catalyzing the oxidation-addition reaction of prochiral compounds, characterized in that:

the prochiral compound is a compound shown as a formula I:

in the formula I, R1 and R2 are independently selected from H, C1-C5 alkyl; a is methylene; n is an integer of 0 to 5;

the prochiral compound is subjected to oxidation reaction with an oxidant under the action of immobilized monoamine oxidase, and then subjected to addition reaction with MR to generate a compound shown in a formula II:

in the formula II, R1 and R2 are independently selected from H, C1-C5 alkyl; a is methylene; n is an integer of 0 to 5;

r is selected from-CN, -SCN, -SO₃Na、-SO₃H；

M is selected from alkali metal, alkaline earth metal and H.

4. The use according to claim 3, characterized in that said use comprises the following steps: under the catalysis of the immobilized monoamine oxidase of claim 1 or the immobilized monoamine oxidase prepared by the method of claim 2, the prochiral compound and MR undergo an asymmetric oxidation-addition reaction with an oxidant in an aqueous solution at pH 6.0-8.0 to form the compound represented by formula II.

5. Use according to claim 4, characterized in that: the dosage of the immobilized monoamine oxidase is 25-250 g/L; the dosage of the MR is 100-1200 mmol/L; the aqueous solution is a buffer solution with the pH value ranging from 6.0 to 8.0, and the concentration of the buffer solution is 0.05 to 0.1 mol/L; the reaction temperature of the oxidation-addition reaction is 20-50 ℃.

6. Use according to any one of claims 3 to 5, characterized in that: the concentration of the prochiral compound in the reaction solution is 100-1000 mmol/L.

7. A method of synthesizing (1S,2R,5R) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-carbonitrile comprising catalyzing an oxidation-substitution reaction of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane with the immobilized monoamine oxidase of claim 1 or prepared by the method of claim 2.

8. The method according to claim 7, wherein the reaction solution contains 100 to 1000 mmol/L6, 6-dimethyl-3-azabicyclo [3.1.0] hexane, 25 to 250g/L recombinant monoamine oxidase or immobilized monoamine oxidase, and 100 to 1200mmol/L MCN, and the reaction is carried out in the presence of an oxidizing agent at a temperature of 20 to 50 ℃; wherein M is selected from alkali metal, alkaline earth metal and H.

9. A process for the synthesis of (1S,2aR,5aR) -octahydrocyclopenta [ c ] pyrrole-1-carbonitrile comprising catalyzing the oxidation-substitution of octahydrocyclopenta [ c ] pyrrole with an immobilized monoamine oxidase of claim 1 or prepared according to the process of claim 2.

10. The method according to claim 9, wherein the reaction solution contains 100 to 1000mmol/L of 5, 5-dimethyl-octahydrocyclopenta [ c ] pyrrole, 25 to 250g/L of recombinant monoamine oxidase or immobilized monoamine oxidase, and 100 to 1200mmol/L of MCN, and the reaction is carried out in the presence of an oxidizing agent at a temperature of 20 to 50 ℃; wherein M is selected from alkali metal, alkaline earth metal and H.